Calpains, the thiol proteinases of the calciumdependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements.

Localization of the two main components of the Ca2+-dependent proteolytic system has been investigated in human neuroblastoma LAN-5 cells. Using a monoclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in two granule-like structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indicating that aggregation of calpastatin is a general property and not an exclusive characteristic of neuronal-like cells. The existence of such calpastatin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounded organelles. Calpastatin distribution is affected by the intracellular increase in free Ca2+, which results in calpastatin progressively becoming a soluble protein. However, calpain is distributed in the soluble cell fraction and, in activating conditions, partially accumulates on the plasma membrane. Similar behaviour has been observed in calpastatin localization in LAN-5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of these cells. The involvement of calpastatin in controlling calpain activity, rather than its activation process, and the utilization of changes in calpastatin localization as a marker of activation of the system is discussed.

We have previously reported that, in neuroblastoma LAN-5 cells, calpastatin is in an aggregated state, close to the cell nucleus [de Tullio, Passalacqua, Averna, Salamino, Melloni and Pontremoli (1999) Biochem. J. 343, 467-472]. In the present paper, we demonstrate that aggregated calpastatin is predominantly in a phosphorylated state. An increase in intracellular free [Ca2+] induces both dephosphorylation of calpastatin, through the action of a phosphoprotein phosphatase, and its redistribution as a soluble inhibitor species. cAMP, but not PMA-induced phosphorylation, reverses calpastatin distribution favouring its aggregation. This intracellular reversible mechanism, regulating the level of cytosolic calpastatin, could be considered a strategy through which calpain can escape calpastatin inhibition, especially during earlier steps of its activation process.

Key words: Proteolysis; Calcium; Calpain; activity and thus it can be considered as an active species of Activation process calpain. Materials and methods I. Introduction Purification of human erythrocyte calpainThe Ca2+-dependent proteinase, calpain, is normally locaHuman erythrocyte calpain was purified as previously described lised in the cytosol of the cells [1][2][3][4][5], together with its natural[15], modified as follows: packed red cells 50 ml were lysed in inhibitor calpastatin. In this cell localisation, calpain is pos-5 vols. of water containing 1 mM EGTA; the membranes were discarded by centrifugation at 25 000 ×g for 10 min and the supernatant tulated to be inactive, due to the high requirement for Ca 2+ was treated with 125 g (wet powder) of DE 32 previously extensively [6][7][8], in the absence of which the enzyme retains a native washed with 50 mM sodium acetate, pH 6.7, containing 0.1 mM conformation with an inaccessible active site. The transition EGTA (buffer A) and stirred for 10 min at 5°C. The resin was colfrom a high to low calcium requiring form results from aulected on a Buckner funnel, washed with 1 1 of buffer A and transferred on a glass column (2.5x 15 cm). The absorbed proteins were toproteolysis, which causes the removal of fragments at the Neluted in a single step with 0.2 M NaC1 dissolved in buffer A. The terminal region of both 80 kDa catalytic and 30 kDa subunits fractions containing calpain activity were collected, precipitated in [9][10][11][12]. The native catalytic subunit is then converted into a 75 50% saturated ammonium sulfate and centrifuged at 25000×g for kDa species, and the small subunit into a 18 kDa fragment.10 min. The pellet was suspended in buffer A and dialyzed for 4 h This new enzyme form expresses full catalytic activity at conin the same buffer solution. The dialyzed material was then loaded onto a column (2.5×3 cm) of Source 15Q (Pharmacia) previously centrations of calcium 50-100-times lower than that required equilibrated in buffer A. The proteins were eluted with a linear graby the native form, and has also been identified in red cells dient of 0~).3 M sodium chloride. The fractions containing calpain enriched with Ca 2+ following exposure of cells to a ionophore, activity were collected, dialyzed for 4 h against sodium borate (50 It seems therefore that autoproteolysis is an essential step that mM, pH 7.5) containing 0.1 mM EGTA (buffer B). To the dialyzed material was added sodium chloride at a final concentration of 0.3 M and the solution loaded onto a column (1 × 10 cm) containing PhenylSepharose CL 4B (Pharmacia) equilibrated with buffer B, containing *Corresponding author. Fax: (39) (10) 354415.0.3 M NaC1. The resin was washed and the bound proteins were 0014-57931961512.00

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90–NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.